The impact of image segmentation on 0.84-ml nominal voxel volume proton spectroscopic imaging in normal brain and in age-related cortical atrophy was investigated. Segmentation improved the gray matter-white matter (GM-WM) contrast for N-acetyl aspartate (NAA)/creatine (Cr) and choline (Cho)/Cr in normal brain, and for NAA/Cho and NAA/Cr in atrophic brain. NAA(GM/WM) (approximately 0.7), Cho(GM/WM) (approximately 0.8), and Cr(GM/WM) (approximately 1.3) in normal brain obtained with segmentation agreed with values obtained with quantitative magnetic resonance spectroscopy. Age-related cortical atrophy led to decreased cortical GM NAA/Cho and NAA/Cr; no changes were evident in WM or in NAA(GM/WM), Cho(GM/WM), or Cr(GM/WM). GM/WM metabolite analysis may be of limited use in conditions in which parallel metabolite changes occur in WM and GM.